Proteases, which catalyze the hydrolysis of amide bonds in peptides and proteins, play essential roles in most biological processes and are important therapeutic targets for a multitude of diseases, including cancer, viral, parasitic, and bacterial infections, inflammation and cardiovascular disease. Indeed, several thousand proteases are predicted to be coded by the human structural gene pool. Upon the complete sequencing of the human genome many new protease coding sequences will be identified by sequence homology as well as by other methods. The biological function of the corresponding proteases must be determined to derive the full value of this gene sequence information. However, how will we be able to rapidly and efficiently establish the function of the huge number of newly identified proteases when complete characterization of the biological function of many heavily studied proteases has not yet been accomplished? In this grant we propose to develop synthetic combinatorial library approaches that could potentially play a huge role in elucidating the function of proteases. In order to realize this potential we propose to achieve to specific aims. First, we propose to develop fluorogenic substrate library approaches to rapidly identify the preferred peptide substrate amino acid sequence of any protease target. The identified substrate specificity profiles will greatly facilitate the elucidation of the natural substrates of proteases. In addition, substrate specificity provides important information in the design of efficient fluorogenic substrates for assays and of potent inhibitors. Second, we propose to develop library strategies to rapidly identify potent, cell permeable inhibitors of proteases. In this grant period we propose to target the aspartyl and cysteine protease classes by preparing small molecule libraries where diverse functionality is displayed about minimal tight binding fragments, or pharmacophores, based upon the mechanism of each of the protease classes. The identified cell permeable, small molecule inhibitors have proven to be powerful "chemical genetic" tools to understand the function of the targeted protease in whole cells and even in animals.